Discovery and utilization of sorghum genes (ma5/ma6)

ABSTRACT

Methods and composition for the production of non-flowering or late flowering sorghum hybrid. For example, in certain aspects methods for use of molecular markers that constitute the Ma5/Ma6 pathway to modulate photoperiod sensitivity are described. The invention allows the production of plants having improved productivity and biomass generation.

This application claims priority to U.S. Provisional Application No. 61/082,388, filed on Jul. 21, 2008. The foregoing application is incorporated herein by reference in its entirety.

This invention was made with government support under grant number DBI-0321578 awarded by the U.S. National Science Foundation and grant number DE-FG02-06ER64306 awarded by the U.S. Department of Energy. The United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of plant genetics and molecular biology. More particularly, it concerns producing high biomass sorghum hybrids by utilizing molecular markers.

2. Description of Related Art

Biomass yield is one of the most important attributes of a biomass or bioenergy crop designed for ligno-cellulosic conversion to biofuels or bioenergy. Growth duration is a primary determinant of biomass yield, therefore late or non-flowering plants accumulate the most biomass assuming environmental conditions allow yield potential to be expressed.

Once grain sorghum initiates flowering, growth of the vegetative plant (stem, leaves) stops so that carbon and nitrogen compounds to be used for grain production. As a consequence, biomass accumulation overall decreases to some extent during the reproductive phase and ceases once grain filling has been completed (unless ratooning follows grain production).

In contrast, a late or non-flowering bioenergy sorghum crop grown for biomass production will continue to accumulate biomass by building larger vegetative plants until frost or adverse environmental conditions inhibit photosynthesis (e.g., drought, cold). It is estimated that late/non-flowering biomass sorghum will generate more than two times the biomass accumulated by grain sorghum per acre assuming reasonable growth conditions throughout the growing season. Therefore, there is a need for producing late or non-flowering sorghum.

SUMMARY OF THE INVENTION

The present invention overcomes a major deficiency in the art in producing high biomass sorghum hybrid by using molecular markers for selection.

In one aspect the invention provides a method for producing a late flowering or non-flowering hybrid sorghum plant comprising crossing a first early flowering sorghum plant with a second early flowering sorghum plant, wherein each of the first and second early flowering sorghum plants is homozygous recessive for at least one allele contributing to an early flowering phenotype, and wherein the first and second early flowering sorghum plants are not homozygous recessive for the same allele contributing to an early flowering phenotype. In one embodiment, the hybrid progeny plant comprises a dominant Ma7 or Ma3 allele.

In a further aspect, the invention provides crossing a first sorghum plant heterozygous dominant for at least a Ma5 or Ma6 allele with a second sorghum plant homozygous recessive for at least the Ma5 or Ma6 allele.

In yet a further aspect the invention provides crossing a first sorghum plant homozygous dominant for at least a Ma5 or Ma6 allele with a second sorghum plant homozygous recessive for at least the Ma5 or Ma6 allele.

In certain embodiments, the first or second early flowering sorghum plant may be produced by a) crossing a late flowering or non-flowering sorghum plant homozygous dominant for Ma5 and Ma6 comprising superior bioenergy properties with an early flowering sorghum plant homozygous recessive for a Ma5 or Ma6 allele; b) inbreeding a F₁ progeny; and c) selecting for an early flowering sorghum F₂ plant homozygous recessive for Ma5 or Ma6 but not homozygous recessive for the same Ma5 or Ma6 allele and comprising said superior bioenergy properties.

In another embodiment, the first or second early flowering sorghum plant may be produced by mutagenizing a late flowering or non-flowering sorghum plant to produce early flowering progeny comprising an inactive gene in a photoperiod sensing pathway. The gene in a photoperiod sensing pathway is selected from the group consisting of Ma3, Ma5, Ma6 and Ma7, in certain embodiments. For instance, the Ma3 gene may comprise a nucleic acid encoding PhyB, the Ma5 gene may comprise a nucleic acid encoding a COP9FUS5 homolog or a Myb-transcription factor, the Ma6 gene may comprise a nucleic acid encoding sorghum Prr37, and the Ma7 gene may comprise a nucleic acid encoding a polypeptide selected from the group consisting of PhyC, a MADS-box 14 protein and AP1.

In some embodiments, the first or second early flowering sorghum is selected from the group consisting of ATx623, EBA-3 and R.07007.

In certain embodiments the invention provides a method of selecting for a progeny plant of the cross according to the invention comprising marker-assisted selection comprising at least a first genetic marker genetically linked to a Ma5 or Ma6 allele. For instance, in one embodiment, the genetic marker genetically linked to the Ma5 allele may comprises a nucleic acid encoding a polypeptide selected from the group consisting of COP9FUS5 homolog and a Myb-transcription factor and in another embodiment, the genetic marker genetically liked to the Ma6 allele comprises a nucleic acid encoding a sorghum Prr37. The sorghum Prr37 polypeptide may comprises a lysine at position 166 or may be encoded by a nucleic acid molecule comprising SEQ ID NO:1, in particular embodiments of the invention.

In further embodiments, genetic markers in accordance with the invention may be linked to a quantitative trait locus (QTL). In some embodiments, the QTL is selected from the group consisting of FlrAvgB1, FlrAvgD1, FlrFstG1, FltQTL-DFG, FltQTL-DFB, QMa50.txs-A, QMa50.txs-C, QMa50.txs-F1, QMa50.txs-F2, QMa50.txs-H, QMa50.txs-I, QMa1.uga-G, QMa1.uga-D, and QMa5.uga-D.

In still further embodiments, genetic markers in accordance with the invention may be selected from the group consisting of sequence variants revealed by direct sequence analysis, restriction fragment length polymorphisms (RFLP), isozyme markers, allele specific hybridization (ASH), amplified variable sequences of plant genome, self-sustained sequence replication, simple sequence repeat (SSR) and arbitrary fragment length polymorphisms (AFLP).

Another aspect of the invention provides harvesting a progeny hybrid plant of the invention to produce biomass, bioenergy, bioproducts or sugar/starch. In yet another aspect, the invention provides a late flowering or non-flowering sorghum hybrid seed produced in accordance with the invention and sorghum hybrid plants grown from the seed.

In a further aspect, the invention provides a method of producing an inbred early flowering sorghum plant comprising: a) crossing a late flowering or non-flowering sorghum plant homozygous dominant for Ma5 and Ma6 with an early flowering sorghum plant homozygous recessive for a Ma5 or Ma6 allele; b) inbreeding the F₁ progeny; and c) selecting for an early flowering sorghum F₂ plant homozygous recessive for Ma5 or Ma6 but not homozygous recessive for the same Ma5 or Ma6 allele. In certain embodiments, the late flowering or non-flowering sorghum plant comprises superior bioenergy properties. In further embodiments, the selected early flowering sorghum F₂ plant comprises said superior bioenergy properties.

In yet a further aspect, the invention provides an inbreed early flowering sorghum seed produced in accordance with the invention and inbreed sorghum plants grown from the seed.

In certain aspects, the invention provides a method of identifying the genotype of a sorghum plant for a Ma5 or Ma6 allele comprising: a) obtaining a sorghum plant; and b) assaying the sorghum plant for a genetic marker genetically linked to the Ma5 or Ma6 allele. In one embodiment, the genetic marker genetically linked to the Ma5 allele may be a nucleic acid encoding a polypeptide selected from the group consisting of COP9FUS5 homolog and a Myb-transcription factor. In another embodiment, the genetic marker genetically linked to an Ma6 allele may be a nucleic acid encoding a sorghum Prr37 polypeptide. In certain embodiments, the sorghum Prr37 polypeptide may comprise a lysine at position 166 or may be encoded by a nucleic acid molecule comprising SEQ ID NO:1.

Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.

As used herein the terms “encode” or “encoding” with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan however these terms may be used interchangeably with “comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A-C: Alignment of EBA3 and RTx436 SbPRR37 cDNA sequences showing DNA sequence differences (bolded).

FIG. 2: Comparison of protein sequences of Prr37 proteins encoded by EBA-3 and RTx436 derived from cDNA sequences. 2.1 corresponds to the sorghum Prr37 protein encoded by EBA-3 (Ma6) and 2.2 corresponds to the sorghum Prr37 protein encoded by RTx436 (ma6). An amino acid difference in the Prr37 protein putative dimerization domain is bolded and underlined; (K (lysine) in EBA-3, N (asparagine) in RTx436. Three additional differences in amino acid sequence are bolded.

FIG. 3: Alignment of partial promoter sequences of SbPRR37 derived from EBA-3 and RTx436. Query refers to EBA3 and Subject refers to BTx623.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention

The instant invention overcomes several major problems with current sorghum production technologies in producing sorghum hybrids that have long duration of vegetative growth due to late flowering or lack of flowering, from inbreds that will flower sufficiently early in regions optimal for hybrid seed production, by manipulation of several QTL and the corresponding genes/alleles that constitute the Ma5/Ma6 pathway that regulates photoperiod sensitivity and flowering time in sorghum. Further embodiments and advantages of the invention are described below.

II. Sorghum

Increased demands on the agricultural and forestry industries due to world population growth, especially recent urgent need in biofuels production, have resulted in efforts to increase plant production and/or size. Sorghum has been an excellent biomass source with its high yield potential, high water use efficiency, and established production systems. Certain embodiments of the present invention disclose methods to generate sorghum genotypes with the genetic potential for improved biomass production.

Sorghum is a genus of numerous species of grasses, some of which are raised for grain and many of which are used as fodder plants either cultivated or as part of pasture. The plants are cultivated in warmer climates worldwide. Species are native to tropical and subtropical regions of all continents in addition to Oceania and Australasia. Sorghum is in the subfamily Panicoideae and the tribe Andropogoneae (the tribe of big bluestem and sugar cane). Sorghum is known as great millet and guinea corn in West Africa, kafir corn in South Africa, dura in Sudan, mtama in eastern Africa, jowar in India and kaoliang in China.

Sorghum is well adapted to growth in hot, arid or semi-arid areas. The many subspecies are divided into four groups—grain sorghums (such as milo), grass sorghums (for pasture and hay), sweet sorghums (formerly called “Guinea corn”, used to produce sorghum syrups), and broom corn (for brooms and brushes). The name “sweet sorghum” is used to identify varieties of Sorghum bicolor that are sweet and juicy. High biomass Sorghum as a source of biofuels has also drawn a lot of attention recently.

Sorghum species contemplated in this invention include, but are not limited to, Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor (primary cultivated species), Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum timorense, Sorghum trichocladum, Sorghum versicolor, Sorghum virgatum, and Sorghum vulgare.

III. Photoperiod Sensitivity

The present invention relates to methods of modulating photoperiod sensitivity and flowering time in sorghum for high biomass production. Photoperiod sensitivity refers to the fact that some plants will not flower until they are exposed to day lengths that are less than a critical photoperiod (short day plants) or greater than a critical photoperiod (long day plants). Long day (LD) and short day (SD) plant designations refer to the day length required to induce flowering. Facultative LD or SD plants are those that show accelerated flowering in LD or SD but will eventually flower regardless of photoperiod. Most plants including sorghum must pass through a juvenile stage (lasting ˜14-21 days for sorghum) before they become sensitive to photoperiod.

Sorghum is a facultative SD plant where long days inhibit flowering and short days accelerate flowering. The degree of photoperiod sensitivity in sorghum refers to the length of the short days that are required to induce flowering. A highly photoperiod sensitive sorghum will require photoperiods less than ˜12 hours before flowering occurs whereas plants with low photoperiod sensitivity only require day lengths less than ˜14 hours to induce flowering. Different sorghum genotypes vary in their degree of photoperiod sensitivity. Sorghum inbreds have been identified with photoperiod sensitivity ranging from ˜10.5 to ˜14 hours and still others that are nearly completely insensitive to photoperiod. For example, in College Station, Tex., photoperiod insensitive sorghum planted in April will flower in approximately 48-55 days. In contrast, highly photoperiod sensitive sorghum hybrids with the Ma5/Ma6 genotype flower in mid to late September in College Station, Tex. (˜175-180 days).

For example, “early flowering sorghum” may be a plant that flowers in 50 to 120 days after planting between April 1 and April 19 in College Station, Tex.; a “late or non-flowering sorghum” may be a plant that flowers 150 to 200 or more days after planting or does not flower under these conditions The number of days to flowering will depend on the planting date and latitude where a sorghum genotype is planted because these factors determine when the plants are exposed to days that are sufficiently short to induce flowering. In general, late flowering photoperiod sensitive plants such as sorghum with the genotype Ma5_Ma6_ will not flower until day lengths are less than 12.2 hrs, whereas less photoperiod sensitive early flowering sorghum with recessive forms of Ma5, and Ma6 (and potentially Ma 7, Ma3, Ma1, etc.) will flower when exposed to day lengths (photoperiods) of ˜12.4-14 hr or longer depending on genotype.

A. Utility of the Ma5/Ma6 System for Bioenergy Sorghum Hybrid Production

Certain aspects of this invention involve the use of the Ma5/Ma6 system to produce early flowering inbreds that when crossed generate high biomass or bioenergy sorghum hybrid seed that can be planted at any time of the year suitable for production, where the hybrid plants will have long growth duration (i.e., late flowering or non-flowering) at all latitudes from ˜40 degrees N/S to the equator (40N=the upper mid-west where sorghum growth is limited by cold). In another aspect, this same flowering control system can also be used to design sweet sorghum hybrids that grow for a specified number of days prior to flowering at different latitudes from early flowering inbreds suitable for hybrid seed production.

Table 1 below describes the relationship between latitude and daylength at planting and harvest for biomass/bioenergy production regions from ˜40 degrees N/S to the equator. At higher latitudes, planting date is later in the year and harvesting occurs earlier due to longer duration of winter and low temperatures (shorter season). At lower latitudes, planting can be done earlier in the year or virtually any time in some locations and harvesting later in the year or multiple times during the year, including times of the year when daylength is less than 12 hours (Table 1).

TABLE 1 Relationship between latitude of crop production and daylength Planting Daylength Harvest Daylength City Latitude date hours date hours DesMoines, IA 41.35N 15-May 14.3 1-Oct 11.6 New York, NY 40.42N 30-May 14.6 1-Oct 11.6 Amarillo, TX 35.05N 15-May 13.8 15-Oct 11.1 College Station, TX 30.37N 20-Mar 11.7 15-Nov 10.4 Beaumont, TX 30.05N 20-Mar 11.8 15-Nov 10.5 Weslaco, TX 26.09N 20-Mar 11.8 1-Dec 10.5 Puerto Rico 18.57N monthly 10.8-13.2 monthly 10.8-13.2 Panama City 08.57N monthly 11.4-12.6 monthly 11.4-12.6 Equator 0 monthly 12 monthly 12 Brazilia, Brazil 16.12S monthly 11.1-12.9 monthly 11.1-12.9 Brisbane, AU 27.30S 20-Sep 11.9 15-Mar 12.2

Sorghum is insensitive to photoperiod during the juvenile phase which lasts for ˜14-21 days post planting depending on genotype. Therefore, bioenergy sorghum hybrids need to have sufficient photoperiod sensitivity to prevent flowering at the daylengths that occur ˜14-21 days post-planting at all latitudes used for bioenergy crop production. In addition, bioenergy sorghum hybrids that are planted in long days that block flowering may also require increased photoperiod sensitivity in order to block flowering prior to frost or harvest if daylengths decrease significantly during the growing season.

Certain aspects of the present invention involves the identification of allelic combinations of Ma5/Ma6 and other genes that repress flowering that work in hybrid combination to block flowering at daylengths as short as 11-10.5 hours. The early flowering inbreds used to produce late/non-flowering hybrid seed are designed to flower early due to different recessive genes that control flowering time. Therefore, when these inbreds are crossed, the F1 hybrids contain dominant genes at all loci involved thereby delaying flowering until plants are exposed to very short photoperiods.

Certain embodiments of the present invention provide sorghum genotypes that contain versions of Ma5 and Ma6 that in combination delay flowering until day lengths are less than 12 hr 20 min (Rooney and Aydin, 1999). There is evidence that additional genes such as Ma1-Ma4 enhance sorghum photoperiod sensitivity. In addition, it is likely that different alleles of Ma5 and Ma6 exist that can be used to make bioenergy sorghum hybrids even more photoperiod sensitive (less than 12 hr) increasing their utility for growing regions closer to the equator where bioenergy sorghum will be planted and grown in day lengths shorter than 12 hours (Table 1). For example, a study by Miller et al. (1968) identified five groups of sorghum that had short day requirements for flowering that ranged from ˜13 hr to ˜11.1 hr. This genetic material, and other genotypes identified in accordance with the present invention, flower late when growing at low latitudes in places such as Puerto Rico. In another study, Craufurd et al. (1999) identified sorghum genotypes with critical photoperiods between 10.2 and 11 hrs. In certain aspects of the invention, these materials have been investigated to identify genes with similar action to Ma5/Ma6 and alleles of Ma5 and Ma6 that would be useful for breeding PS hybrids for use over the entire range of latitudes from 40N/S to the equator.

B. Genetic Pathway of Photoperiod Sensitivity and Uses Thereof

Photoperiod sensitivity and late flowering is mediated in sorghum and rice by genes that repress activation of FT (flowering locus T) and AP1 and the transition of the apex from vegetative growth to forming reproductive structures. The repressors of flowering in sorghum act in a dominant fashion. The repressors are inactivated or less active under short photoperiods (and thermal periods). The vegetative or non-flowering state is maintained in part by light mediated signaling through PhyB and PhyC and possibly from other sources (PhyA, etc.) and partly by output from a circadian clock. The light signaling pathway involves a series of steps and genes, some of which may act directly to repress FT, and others of which act downstream from the circadian clock through modulation of homologs of GI, CO, and other genes that modulate repression of FT.

The repressing pathway can be inactivated by disrupting the function of any of the genes that are in the signaling pathway (PHYB, PHYC, or a gene between the photoreceptors and FT, and genes involved in clock function or input/output). The disruption of a gene in the flower repression pathway converts a photoperiod sensitive genotype into a less photoperiod sensitive genotype or photoperiod insensitive genotype that will flower early or at longer day lengths. If genotypes that are photoperiod insensitive due to inactivation of different genes in the flowering repression pathway are crossed, then the hybrid will be photoperiod sensitive and later flowering because active alleles contributed by the gametes from each line complement inactive alleles present in the gametes/genome of the other parental inbred line.

IV. Production of Photoperiod Sensitive Hybrid Using Ma5/Ma6 System

In certain aspects of the present invention, early flowering inbred sorghum genotypes with the proper allelic combinations of Ma5 and Ma6 can be crossed to produce photoperiod sensitive late-flowering sorghum hybrids (Ma5_Ma6_) ideal for biomass/bioenergy production with the use of molecular markers. In one embodiment, the early flowering photoperiod insensitive sorghum inbreds contain complementary pairs of dominant/recessive Ma5/Ma6 genes (Ma5ma6 and ma5Ma6 respectively).

One advantage of the Ma5/Ma6 system is the ability of this system to produce sorghum hybrids that have long duration of vegetative growth due to late flowering or lack of flowering, from inbreds that will flower sufficiently early in regions optimal for hybrid seed production (such as high plains of Texas).

The production of bioenergy sorghum hybrids is also important because hybrids are preferred commercially due to hybrid vigor that generates greater yield, and the ability to better control seed stocks through hybrid seed production. The increase in yield attributed to hybrid vigor in sorghum is typically ˜20% to ˜50%. Photoperiod sensitive bioenergy sorghum hybrids that flower late or that do not flower are important for bioenergy production for several reasons: long duration of vegetative growth associated with late/non-flowering genotypes increases biomass yield per acre, high levels of photoperiod sensitivity will allow nearly year round planting of bioenergy sorghum hybrids at lower latitudes, and plants growing vegetatively (non-flowering) are more drought tolerant than plants that are in the reproductive phase of development; this is an important attribute of bioenergy sorghum.

A. Breeding Material and Methods

In further aspects of the present invention, naturally occurring alleles of Ma5 and Ma6 as well as other maturity genes (e.g., PHYB, PHYC) that are involved in the photoperiod-sensing pathway can be used to construct early flowering inbreds that can be crossed to produce late flowering hybrids.

In one embodiment, sorghum line R.07007 or EBA-3 is a primary source of both ma5 (recessive form) and Ma6 (dominant form), although other versions of Ma6 derived from photoperiod sensitive sorghum accessions may also be utilized. In another embodiment, dominant forms of Ma5 are derived from grain sorghum female lines that may be used for hybrid seed production.

In addition to working with naturally occurring genetic variants, certain embodiments of the present invention comprise mutagenizing any group of PS genotypes and identify PI lines derived from the parental lines that contain an inactive gene in the pathway that represses flowering. Crossing photoperiod insensitive early flowering genotypes that contain different inactive genes in the pathway that controls flowering time will generate photoperiod sensitive late flowering hybrids.

An exemplary approach involves screening photoperiod sensitive (late flowering) sorghum germplasm for accessions that express superior bioenergy traits. These accessions (most likely Ma5/Ma6) are then crossed to R.07007 or EBA-3 (ma5ma5ma7ma7Ma6Ma6). F2 progeny from these crosses that flower early (ma5ma5) but that retain Ma6Ma6 are selected by phenotyping and marker-assisted selection. The resulting early flowering inbreds (ma5ma5Ma6Ma6) can then be crossed with elite grain female A-lines that have the genotype (Ma5Ma5Ma7Ma7ma6ma6), to produce bioenergy hybrids that are Ma5_Ma7_Ma6_ that will flower late.

B. Sorghum Mutagenesis

In another aspect of the invention, mutagenesis of late flowering sorghum genotypes to create early flowering genotypes could be carried out in the following exemplary manner. The seed from a late flowering sorghum inbred would be germinated and treated with a mutagen such as EMS (ethyl methanesulphonate) or ENU (1-ethyl-1-nitrosourea) or using X-rays or neutron bombardment to induce changes in DNA sequence throughout the sorghum genomes of thousands of seedlings. The M1 seedlings (M1 refers to the first generation of plants that were exposed to a mutagen) surviving the treatment would be grown to maturity and self-pollinated. M2 seed derived from a large number of M1 plants would be grown out and screened for M2 plants that flower early under conditions where the parental inbred flowers late. An early flowering phenotype would be consistent with mutation in a gene that represses flowering such as Ma5 or Ma6.

C. Molecular Markers

a. Marker Assisted Selection

Marker assisted selection or marker aided selection (MAS) is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g., productivity, disease resistance, abiotic stress tolerance, and/or quality). This process has been used in plant breeding.

Considerable developments in biotechnology have led plant breeders to develop DNA marker aided selection systems to augment traditional phenotypic-pedigree-based selection systems. Marker assisted selection (MAS) is an indirect selection process where a trait of interest is selected not based on the trait itself but on a marker linked to the gene (allele) that controls expression of the trait. For example if MAS is being used to select individuals with disease resistance, then a marker allele which is linked to the gene conferring disease resistance is scored or selected for rather than disease resistance per se. The assumption is that the marker allele is associated with the gene and/or quantitative trait locus (QTL) of interest that confers the trait under selection. MAS can be useful to select for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development.

In certain embodiments, a marker may be;

Morphological—First marker loci available that have obvious impact on morphology of plant. Genes that affect form, coloration, male sterility or resistance among others have been analyzed in many plant species. Examples of this type of marker may include the presence or absence of awn, leaf sheath coloration, height, grain color, aroma, etc.

Biochemical—A gene that encodes a protein that can be extracted and observed; for example, isozymes and storage proteins.

Cytological—The chromosomal banding produced by different stains; for example, G banding.

Biological—Different pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites.

DNA-based and/or molecular—A unique (DNA sequence), occurring in proximity to or within the gene or locus of interest, can be identified by a range of molecular techniques such as direct sequencing, RFLPs, RAPDs, AFLP, DAF, SCARs, microsatellites, etc. DNA markers detect variation in DNA sequence, or DNA polymorphisms, that distinguish individuals. DNA polymorphisms include differences in single nucleotide sequences (SNPs), simple sequence repeats (SSRs), inversions or deletions (INDELS). DNA markers are designed to identify DNA sequence differences by one of several methods including; direct sequence analysis, electrophoretic separation of DNA fragment sizes following digestion of genomic DNA with restriction enzymes (RFLP) or after DNA amplification using PCR (AFLP, SSRs), or based on differences in amplification or probe hybridization (microarrays, Taqman probes, etc.).

As used herein, an “inherited genetic marker” is an allele at a single locus. A locus is a position on a chromosome, and allele refers to conditions of genes; that is, different nucleotide sequences, at those loci. The marker allelic composition of each locus can be either homozygous or heterozygous.

Coinheritance, or “genetic linkage,” of a particular trait and a marker suggests that they are physically close together on the chromosome. Linkage is determined by analyzing the pattern of inheritance of a gene and a marker in a cross. The unit of recombination is the centimorgan (cM). Two markers are one centimorgan apart if they recombine in meiosis once in every 100 opportunities that they have to do so. The centimorgan is a genetic measure, not a physical one. Those markers located less then 50 cM from a second locus are said to be genetically linked, because they are not inherited independently of one another. Thus, the percent of recombination observed between the loci per generation will be less than 50%. In particular embodiments of the invention, markers may be used located less than about 45, 35, 25, 15, 10, 5, 4, 3, 2, or 1 or less cM apart on a chromosome.

The gene of interest is directly related with production of protein(s) or RNAs (e.g., miRNA) that produce certain phenotypes whereas markers should not influence the trait of interest but are genetically linked to an allelic form of a gene that modifies a trait (and so the marker and gene remain together during segregation of gametes due to the physical linkage between marker and gene, and a reduction in homologous recombination between the marker and gene of interest due to their close proximity on a strand of DNA). In many traits genes are discovered and can be directly assayed for their presence with a high level of confidence. However, if a gene is not isolated, marker's help is taken to tag a gene of interest. In such case there may be some false positive results due to recombination between marker of interest and gene (or QTL). A preferred marker that corresponds to or detects the difference in DNA sequence causing the desired phenotype or trait would elicit no false positive results.

In MAS, generally the first step is to map the gene or quantitative trait locus (QTL) of interest first by using one or more genetic mapping techniques and then use this information to identify DNA markers linked to and flanking the QTL useful for marker-assisted selection. Generally, the markers to be used should be close to the gene of interest (<5 recombination unit or cM) in order to ensure that only a minor fraction of the selected individuals will have a recombination between the DNA marker and target gene following any given cross or meiosis (specifically the DNA sequence variation within the target gene that causes the desired trait). Generally, not only a single marker but rather two markers are used that flank the target gene or QTL as closely as possible in order to reduce the chances of an error due to homologous recombination.

In plants QTL mapping is generally achieved using bi-parental cross populations involving two parents that have a contrasting phenotype for the trait of interest. Commonly used populations are recombinant inbred lines (RILs), doubled haploids (DH), back cross and F2. Linkage between the phenotype and markers that have already been mapped is tested in these populations in order to determine the position of the QTL on the overall genetic map. Such techniques are based on linkage and are therefore referred to as “linkage mapping”.

In contrast to two-step QTL mapping and MAS, a single-step method for breeding typical plant populations has been developed (Rosyara et al., 2007). In such an approach, in the first few breeding cycles, markers linked to the trait of interest are identified by QTL mapping and later the same information in used in the same population. In this approach, pedigree structure is created from families that are created by crossing a number of parents (in three-way or four way crosses). Phenotyping is carried out and genotyping is done using molecular markers mapped the possible location of QTL of interest. This will identify markers and their favorable alleles. Once these favorable marker alleles are identified, the frequency of such alleles will be increased and response to marker-assisted selection is estimated. Marker allele(s) with desirable effect will be further used in next selection cycle or other experiments.

Recently high-throughput genotyping techniques are developed which allows marker aided screening of many genotypes. This will help breeders in shifting traditional breeding to marker-aided selection. One example of such automation is using DNA isolation robots and pipetting robots. A recent example of a high throughput DNA sequencer is the Illumina SGAII or ABI SOLID System.

Genetic markers and QTLs used in certain embodiments of the invention have been disclosed below.

In certain embodiments of the invention, molecular markers are developed that are polymorphic in parental lines of a cross or population, and linked to and flank the Ma5 and Ma6 QTL targeted for marker assisted selection (MAS). The molecular markers in some cases can correspond to and detect specific DNA sequence variants causing dominant or recessive gene action. In certain aspects, DNA may be extracted from the parental sorghum lines and progeny of a cross (F1, F2, backcross, testcross, RIL, etc.) and analyzed with molecular markers for the presence or absence of marker alleles linked to and flanking regions of the genome encoding dominant or recessive forms of Ma5 and/or Ma6. While any molecular marker assay technology could be used, biallelic (or multiallelic) marker assays such as SSRs, or assays such as direct sequencing that detect SNPs/indels are preferred.

b. Ma Genes

There are six classic maturity genes in sorghum that control flowering time termed Ma1-Ma6. Ma1, Ma2, Ma3 and Ma4 were identified by Quinby and his colleagues (Quinby and Karper, 1946; Quinby, 1966; Quinby, 1974). These loci/genes are part of a pathway that inhibits flowering. Therefore in general, sorghum plants with recessive Ma1-Ma6 genes (with low or no activity) flower earlier than plants with dominant or active Ma1-Ma6 genes that repress flowering. Sorghum plants that are Ma1Ma2Ma3Ma4 but recessive at either Ma5 or Ma6 will flower in ˜74 days in College Station, Tex. when planted on April 19 (Rooney and Aydin, 1999) or in ˜85 days when planted on June 1 in Plainview, Tex. (Quinby, 1974). Plants with recessive genes at Ma1-Ma4 (and recessive at Ma5 or Ma6) will flower in ˜48-55 days post planting in these same locations. Ma5 and Ma6 are an additional pair of maturity loci that delay flowering when sorghum is planted ˜April 19 in College Station, Tex. for ˜175 days (mid-late September when photoperiods decrease below 12 h 20 min) (Rooney and Aydin, 1999). Based on information described in more detail below, it is predicted that late flowering Ma5/Ma6 plants also require an active PHYB gene (Ma3)

If an active form of PHYB (or Ma3) is required for Ma5/Ma6 genotypes to express photoperiod sensitivity and flower late, then complementary dominant/recessive forms of Ma3 could also be used to modulate differential flowering time in certain types of inbreds and hybrids. In this case, an early flowering inbred sorghum line that has the genotype ma3ma3Ma5Ma5Ma6Ma6 could be crossed to a second early flowering inbred sorghum genotype that has the genotype Ma3Ma3Ma5Ma5ma6ma6 in order to produce late flowering sorghum hybrids with the genotype Ma3ma3Ma5Ma5Ma6ma6.

Table 2 shows that information about the genetic map location of Ma1 and Ma3 has been published (Klein et al., 2008; Childs et al., 1997). Ma3 encodes the red light photoreceptor phytochrome B that is known to mediate repression of flowering in short day and long day plants (Childs et al., 1998). In addition, the inventors have collected information over the past several years on the genetic map locations of Ma5 and Ma1, loci required in combination with Ma6 to delay flowering ˜175 days in College Station. Ma6 has also been mapped, as well as a modifier of Ma6 activity.

TABLE 2 Sorghum maturity (Ma) genes Locus Map location Gene Reference Ma1 SBI06, ~11-21cM Unknown Klein et al., 2008 Ma2 Unknown Ma3 SBI01, ~166cM PHYB Childs et al., 1998 Ma4 Unknown Ma5 SBI02, ~145-148cM Ma7 SBI01, ~23-26cM Ma6 SBI06, ~11-19cM

b. Sorghum Flowering Time QTL

QTL (quantitative trait loci), quantitative trait inheritance or polygenic inheritance refers to the inheritance of a trait or phenotype that varies in degree of trait expression due to the interactions between two or more genes and the environment. QTL are genetic loci that span regions of a genome that encode genes that contribute to quantitative inheritance of a trait. The contributions of allelic forms of genes that contribute to quantitative traits and the genetic map locations of QTL can be characterized by analysis of populations derived by crossing parental lines that contain different allelic forms of genes that contribute to quantitative trait inheritance.

QTL mapping involves the genetic study of inheritance of alleles that occur in two or more loci and the phenotypes (physical forms or traits) that they produce. Because most traits of interest are governed by more than one gene, defining and studying the entire suite of genes and their alleles that modulate a trait provides an understanding of what effect the genotype of an individual has on the phenotype of that individual.

Genetic analysis involving statistical assessment is required to analyze the interaction of genes and to determine whether they produce a significant effect on the phenotype. QTL identify regions of the genome as containing allelic variation for one or more genes (or regulatory elements) that modulate the trait being assayed or measured. They are shown as intervals spanning a region of a chromosome, genetic map, or DNA sequence, where the probability of association is plotted for each marker used in the mapping experiment.

The QTL techniques were developed in the late 1980s and can be performed on populations of any species. To begin, a set of genetic markers must be developed for the species in question. A marker is an identifiable region of variable DNA sequence (single nucleotide or repeat variation, or inversions/deletions). Biologists are interested in understanding the genetic basis of phenotypes (i.e., physical traits). The aim is to find a marker that is significantly more likely to co-occur (co-segregate following a cross) with the trait than expected by chance, that is, a marker that has a statistically significant association with the trait. It is ideal to identify the specific gene or genes that modulate the trait in question, but this often requires a great deal of time and effort. Instead, they can more readily find regions of DNA that are very close to the genes in question. When a QTL is mapped, it identifies a region of the genome that spans the actual gene underlying the phenotypic trait although the region identified may also encode many genes that do not modulate the target trait.

For organisms whose genomes are known, one might now try to exclude genes in the identified region whose function is known with some certainty not to be connected with the trait in question. If the genome is not available, it may be an option to sequence the identified region and determine the putative functions of genes by their similarity to genes with known function, usually in other genomes.

Another interest of statistical geneticists using QTL mapping is to determine the complexity of the genetic architecture underlying a phenotypic trait. For example, they may be interested in knowing whether a phenotype is modulated by many independent loci, or by a few loci, and do those loci interact. This can provide information on how expression of the phenotype is regulated.

Numerous QTL that modulate flowering time in sorghum have been identified in various studies (e.g., Lin et al., 1995, Paterson et al., 1995, Crasta et al., 1999, Hart et al., 2001; Feltus et al., 2006). The correspondence between QTL that modulate flowering time identified in genetic mapping studies and Ma1-Ma6 is not entirely clear because the location of Ma2 and Ma4 on the sorghum genetic map is not known. Information on various QTL for flowering time in sorghum is listed in Table 3.

TABLE 3 Sorghum flowering time QTL Lin et al., 1995, Paterson et al., 1995; BTx623 X S. propinquum Locus Map location Marker FlrAvgB1 SBI02, ~102-119cM UMC5, UMC139 FlrAvgD1 SBI06, ~9-21cM FlrFstG1 SBI09, ~129-150cM UMC132 Crasta et al., 1999; B35 X RTx430 Locus Map location Gene FltQTL-DFG SBI10, ~70-74cM UMC21 FltQTL-DFB SBI01, ~45cM UMC27, ~10cM from PHY A Hart et al., 2001 (see map positions in Feltus et al., 2006 below) Feltus et al, 2006; summary of QTL from BTx623/IS3620C; BTX623/S. propinquum Locus Map location Marker QMa50.txs-A SBI01, ~182-186cM Xgap36 QMa50.txs-C SBI03, ~40cM Xumc16 - Xtxs422 QMa50.txs-F1 SBI09, ~143cM Xcdo393 QMa50.txs-F2 SBI09, ~143cM Xcdo393 QMa50.txs-H SBI08, ~130-136cM Xtxp105-Xtxs1294 QMaSO.txs-I SBI06, ~10-36cM Xumc119-Xcdo718 Lin et al. (1995), Paterson et al. (l995) Locus Map location Marker QMa1.uga-G SBI09, ~129-150cM Xumc132-pSB445 QMa1.uga-D SBI06, ~31-59cM data requires further analysis QMa5.uga-D SBI06, ~8-20cM tiller flowering

The relationship between Ma6 and Ma1 is uncertain at this time. The impact of Ma1 and Ma6 is quite different, but both QTL map to a similar region on SBI06 making it formally possible that Ma1 and Ma6 are different alleles of the same gene or different genes that reside in the same region of the genome.

Feltus et al. (2006) reported a flowering time QTL (QMa5.uga-D) that controls tiller flowering time that overlaps the region spanned by Ma1 and Ma6. It is formally possible that QMa5.ugaD corresponds to a different allele of Ma1 or Ma6 or a different flowering time gene.

Lin et al. (1995) mapped a flowering time QTL (FlrAvgD1=QMa1.ugaD) on SBI06 (31-59 cM) and suggested that this QTL could correspond to Ma1. Klein et al. (2008) using genotypes known to segregate for Ma1 showed that Ma1 mapped to an adjacent region on SBI06 (˜11-21 cM). The data in Lin et al. (1995) are inconsistent with the assigned map location of QMa1.ugaD in Feltus et al. (2006). Data in Lin et al. (1995) show that QMa1.ugaD maps to the same location as QMa5.ugaD (Feltus et al., 2006).

V. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Methods for Using Marker-Assisted Selection of Sorghum Inbreds that for Production of Photoperiod Sensitive Late or Non-Flowering Sorghum Hybrids

In one non-limiting embodiment of the invention, molecular markers may be used to help convert a photoperiod sensitive (PS) late flowering inbred sorghum (A) that has the genotype Ma5Ma5Ma6Ma6 into a photoperiod insensitive (PI) early flowering inbred that can be used (crossed) in temperate regions to produce sorghum hybrid seed and hybrids that flower late. This can be done as follows:

a. Cross PS sorghum (A) with the genotype Ma5Ma5Ma6Ma6 to a PI sorghum (B) with the genotype ma5ma5Ma6Ma6 to generate an F1 plant.

b. Self the F1 plant and grow out F2 progeny.

c. Use DNA markers to identify progeny (C) that have the genotype ma5ma5Ma6Ma6.

d. The ma5ma5 alleles in (C) will be derived from (B).

e. The Ma6Ma6 alleles in (C) may be derived from either (A) or (B). Selection for the source of Ma6 allele may be important depending on the relative activity of the Ma6 alleles derived from (A) and (B).

f. Cross progeny with the genotype ma5ma5Ma6Ma6 (C) to an elite PI early flowering sorghum (D) with the genotype Ma5Ma5ma6ma6 to produce F1 seed.

g. F1 hybrid plants derived from this cross will be photoperiod sensitive and late or non-flowering with the genotype Ma5ma5Ma6ma6.

In another aspect, the method described above that starts with PS late flowering plants with the genotype Ma5Ma5Ma6Ma6 could involve several alternatives:

a. PI sorghum (B) used above could have the genotype ma5ma5ma6ma6.

In this case, progeny (C) identified by markers with the genotype ma5ma5Ma6Ma6 would have derived ma5 alleles from (B) and Ma6 alleles from (A).

b. PS sorghum (B) could have the genotype ma5ma5ma7ma7Ma6Ma6.

-   -   i. This is a case where recessive alleles in two different genes         with Ma5-like action are needed to make progeny (C) PI, early         flowering, and useful for the generation of PS sorghum hybrids.     -   ii. In this case, DNA markers would be used to identify         progeny (C) that have the genotype ma5ma5ma7ma7Ma6Ma6.     -   iii. In this case, PI progeny (C) with the genotype         ma5ma5ma7ma7Ma6Ma6 could be crossed to an elite PI line (D) with         the genotype Ma5Ma5Ma7Ma7ma6ma6 to produce PS late or         non-flowering sorghum hybrid seed/plants with the genotype         Ma5ma5Ma7ma7Ma6ma6.

In a further aspects, inventors may want to convert a PI early flowering plant that is not suitable for use in the production of PS late or non-flowering sorghum hybrids into a PI early flowering plant that can be used for this purpose. This can be done as follows:

a. Cross a PI early flowering genotype (E) with the genotype ma5ma5ma6ma6 or Ma5Ma5ma6ma6 with a PI early flowering genotype (F) with the genotype ma5ma5Ma6Ma6.

b. Self the resulting F1 plants and use molecular markers to identify progeny (G) with the following genotype; ma5ma5Ma6Ma6.

c. The ma5ma5 alleles could be derived from (E) or (F) depending on the cross involved, whereas the Ma6Ma6 alleles will be derived from (F).

d. Cross progeny (G) to an elite sorghum with the genotype Ma5Ma5ma6ma6 to generate F1 seed/hybrid plants that are PS late or non-flowering.

Example 2 Genetic Map Analysis of Ma5

The location of Ma5, Ma7 and Ma6 on the sorghum genetic map was determined as described below thus enabling the development of DNA markers flanking these loci for use in marker-assisted breeding. The coordinates of Ma5, Ma7 and Ma6 on the TAMU sorghum genetic map (cM) and on the DOE sorghum genome sequence (bp) are listed below: (i) Ma5 QTL coordinates on SBI-02: From 59L10, 67923811 bp, 146.1-148.9 cM to txp428, 68393290 bp, 148.9-152.1 cM; (ii) Ma7 QTL coordinates on SBI-01: From txp208, 6545866 bp, 23.4 cM to txp523, 8017655 bp, 26.5-29.5 cM.

Populations segregating for Ma5/ma5 were constructed by crossing EBA-3 (ma5ma5Ma6Ma6) to A3RTx436 (Ma5Ma5ma6ma6) creating an F1 hybrid that was backcrossed to EBA-3 to create a BC1F1 mapping population that was expected to segregate 1:1 for alleles at the Ma5 locus (Ma5ma5Ma6_; ma5ma5Ma6_). Phenotypic analysis of flowering time of BC1F1 progeny was performed from this cross.

A large population of ˜4200 BC1F1 plants was grown at two locations in College Station, Tex. and was assayed for days to flowering at approximately weekly intervals. The parents of this population flowered between 60-90 days, and the F1 flowered at ˜170-180 days. Data on time to flowering was collected from 2915 plants, whereas the remaining plants either died during growth (a small number of plants) or had not flowered by November when frost terminated their development. Approximately 28% of the population flowered early (before August 7) and there was a period from 105-148 days post planting where fewer plants flowered before a second large cohort of plants initiated flowering. Approximately 72% of the plants flowered after August 7, with many plants flowering well after F1 hybrids flowered at approximately 175 days (more than 220 days). This result indicated that more than one gene with Ma5-like action was segregating in this population based on deviation from 1:1 segregation of PI:PS phenotypes and transgressive segregation for late flowering.

A form of bulk segregant analysis and SSR and AFLP markers were used to map the location of one locus with Ma5 action to a ˜10 cM region on LG-02. This locus was designated Ma5. The location of Ma5 was further refined to a region ˜250,000 bp. Information on the segregation of Ma5 and flowering phenotypes was used to map a second locus with Ma5 like action to LG-01. This locus was designated Ma7. The gene for Ma7 was further fine mapped to a region spanning ˜400,000 bp. Portions of the sorghum genome sequence released by DOE to each of these regions and identified putative genes encoded by these regions based on BLAST analysis and comparison to the colinear region of the rice genome were identified and aligned.

The Ma5 and Ma7 loci were examined and candidate genes in these regions were identified that could explain the observed regulation of flowering time. In the Ma5 locus, a gene homologous to COP9FUS5 was identified as a candidate gene. COP9FUS5 is a subunit of a large signalasome complex (CSN complex) that was initially identified in Arabidopsis as involved in the repression of photomorphogenesis and a range of other activities. This complex acts by targeting transcription factors for degradation that mediate light activated events (such as de-etiolation, light activated gene expression). Therefore, it was reasoned that variation in the activity of COP9FUS5 could modulate flowering time in sorghum by modulating light dependent repression of flowering mediated by the PhyB and PhyC photoreceptors, or by modulating light dependent output from the circadian clock. A gene encoding a Myb-transcription factor that could be involved in flowering time was also identified in the Ma5 fine mapping interval. Myb-transcription factors such as CCA1/LHY (Arabidopsis) are a central part of the circadian clock and allelic variation in this type of gene can modulate flowering time.

Several candidate genes were identified in the Ma7 locus including PhyC, a MADS-box 14 gene and a MADS-box gene corresponding to AP1. AP1 activates meristem identity genes that are involved in the production of floral organs. AP1 is activated by FT in the apex. FT encodes a transmissible protein that travels from the leaf to the apex when photoperiod and other requirements are met such that FT expression is activated. MADS-box 14 is involved in flowering time control in rice so it is also a candidate gene for Ma7. PhyC is also a reasonable candidate for Ma7 because in rice, and presumably sorghum, inactivation of PhyC decreases repression of flowering in long days resulting in early flowering (as observed in EBA-3).

Example 3 Genetic Map Location and Molecular Description of Ma6

Ma6 was mapped in a BC1F1 population created by crossing EBA-3 (ma5ma5Ma6Ma6) to ATx623 (Ma5Ma5ma6ma6), where the F1 derived from this cross was backcrossed to ATx623. Progeny from the BC1F1 population were expected to segregate for the Ma6 locus in a 1:1 ratio (Ma5_ma6ma6 vs. Ma5_Ma6ma6). Late flowering plants from this population were expected to contain the EBA-3 dominant version of Ma6. Genetic mapping initially located Ma6 to an interval on SBI06 spanning from ˜8 cM to ˜21 cM (Ma6 QTL coordinates on LG-06: txp658, 39379760 bp, 8.0-9.9 cM to txp434, 42610705 bp, 17.4-20.7 cM; by coordinates are derived from the DOE pseudomolecule sequence). Further fine mapping narrowed the Ma6 locus to a region spanning from Xtxp598 to a DNA polymorphism present in a DNA binding protein upstream from Ma6. There are approximately ˜20 annotated genes (excluding genes associated with transposons) in this delimited region.

A sorghum gene encoding a homolog of Arabidopsis PRR7 (and rice OsPRR37) was present among the ˜20 genes in the delimited Ma6 locus. The PRR7/PRR37 gene homologs are known to modulate flowering time in several plant species (Arabidopsis, rice, barley) suggesting that the sorghum PRR7/37 gene homolog in the Ma6 locus is likely to be the gene causing differences in flowering time in sorghum. Therefore, cDNA derived from this gene was sequenced from EBA-3 (Ma6) and RTx436 (ma6) and compared (FIG. 1A-C; SEQ ID NOs: 1 and 2). The alignment of the cDNA sequences revealed 5 sequence polymorphisms (FIG. 1A-C). One of these sequence differences (a G (EBA3) to T (RTx436) substitution) caused amino acid 166 to change from a lysine (EBA3) to an asparagine in RTx436 (FIG. 2). This amino acid change is conservative (no charge change) but occurs in a three amino acid sequence of the Prr protein predicted to be involved in dimerization. Therefore it is possible that this change in amino acid sequence alters protein-protein interaction required for normal function of the SbPrr37 protein.

The protein encoded by sorghum PRR37 (Ma6) is homologous to and similar in amino acid sequence to the protein encoded by rice PRR37. The rice Prr37 protein sequence is shown below, where the putative signal receiver domain is shown in bold and the putative dimerization domain (amino acids 166-168) is shown in bold and underlined (amino acid sequence KPI (lysine-proline-isoleucine). The sorghum Prr37 putative dimerization domain of EBA-3 (Ma6) has the sequence KPI (SEQ ID NO:3) identical to rice Prr37 (SEQ ID NO:5) (KPI at positions 166-168), whereas the putative dimerization domain of Prr37 from RTx436 (ma6) (SEQ ID NO:4) has the sequence NPI.

Rice Prr7 Sequence:

  1 mmgtahhnqt agsalgvgvg dandavpgag gggysdpdgg pisgvqrppq vcwerfiqkk  61 tikvllvdsd dstrqvvsal lrhcmyevip aengqqawty ledmqnsidl vltevvmpgv 121 sgisllsrim nhnicknipv immssndamg tvfkclskga vdflv kpi rk nelknlwqhv 181 wrrchsssgs gsesgiqtqk caksksgdes nnnngsnddd dddgvimgln ardgsdngsg 241 tqaqsswtkr aveidspqam spdqladppd stcaqvihlk sdicsnrwlp ctsnknskkq 301 ketnddfkgk dleigsprnl ntayqsspne rsikptdrrn eyplqnnske aamenleess 361 vraadligsm aknmdaqqaa raanapncss kvpegkdknr dnimpslels lkrsrstgdg 421 anaiqeeqrn vlrrsdlsaf tryhtpvasn qggtgfmgsc slhdnsseam ktdsaynmks 481 nsdaapikqg sngssnnndm gsttknvvtk pstnkervms psavkanght safhpaqhwt 541 spanttgkek tdevannaak raqpgevqsn lvqhprpilh yvhfdvsren ggsgapqcgs 601 snvfdppveg haanygvngs nsgsnngsng qngsttavda erpnmeiang tinksgpggg 661 ngsgsgsgnd mylkrftqre hrvaavikfr qkrkernfgk kvryqsrkrl aeqrprvrgq 721 fvrqavqdqq qqgggreaaa dr

Rice Prr7 Protein Features: Location/Qualifiers

  source   1..742 /organism=“Oryza sativa Japonica Group” /cultivar=“Nipponbare” /db_xref=“taxon:39947”   Protein   1..742 /product=“pseudo-response regulator 37”   Region   65..180 /region_name=“REC” /note=“Signal receiver domain; originally thought to be unique to bacteria (CheY, OmpR, NtrC, and PhoB), now recently identified in eukaroytes ETR1 Arabidopsis thaliana; this domain receives the signal from the sensor partner in a two-component systems; contains; cd00156” /db_xref=“CDD:29071”   Site   order(68..69,114,122,144,163,166..167) /site_type=“active” /db_xref=“CDD:29071”   Site   114 /site_type=“phosphorylation” /db_xref=“CDD:29071”   Site   order(117..118,120..122) /site_type=“other” /note=“intermolecular recognition site” /db_xref=“CDD:29071”   Site   166..168 /site_type=“other” /note=“dimerization interface” /db_xref=“CDD:29071”   Region   682..718 /region_name=“CCT” /note=“CCT motif. This short motif is found in a number of plant proteins. It is rich in basic amino acids and has been called a CCT motif after Co, Col and Toc1; pfam06203” /db_xref=“CDD:87043”   CDS   1..742 /gene=“OsPRR37” /coded_by=“AB189039.1:1..2229”

A difference in the expression (gene regulation) of PRR37 in EBA-3 and RTx436 could cause a difference in gene activity corresponding to Ma6 vs. ma6. Preliminary assays showed that PRR37 was expressed differently in EBA-3 and RTx436. Therefore, ˜800 bp of the promoter regions of PRR37 from EBA-3 and RTx436 was sequenced and aligned (FIG. 3). This revealed many sequence differences including several large deletions/insertions in the promoter regions of PRR37 in RTx436 compared to EBA-3 (FIG. 3). These differences in sequence may alter the expression of the PRR37 alleles and contribute to a difference in flowering phenotype.

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

-   Childs et al., Plant Physiol., 116(3):1003-1011, 1998. -   Childs et al., Plant Physiol., 113:611-619, 1997. -   Crasta et al., Mol. Gen. Genet., 262(3):579-588, 1999. -   Craufurd et al., Theor. Appl. Genet., 99:900-911, 1999. -   Feltus et al., Theor. Appl. Genet., 112(7):1295-1305, 2006. -   Hart et al., Theor. Appl. Genet., 103: 1222-1242, 2001 -   Ishikawa et al., Plant Cell, 17(12):3326-3336, 2005. -   Kaczorowski and Quail, Plant Cell, 15(11):2654-2665, 2003. -   Klein et al., Plant Genome, 48: S12-22, 2008 -   Lin et al., Genetics, 141(1):391-411, 1995. -   McClung, Proc. Natl. Acad. Sci. USA, 103(32):11819-11820, 2006. -   Miller et al., Crop Science, 8:499-502, 1968. -   Nakamichi et al., Plant Cell Physiol., 48(6):822-832, 2007. -   Paterson et al., Proc. Natl. Acad. Sci. USA, 92(13):6127-6131, 1995. -   Quinby and Karper, Amer. J. Botany, 33(9):716-721, 1946. -   Quinby, J. R., Crop Science 6:516-518, 1966 -   Quinby, J. R. (1974) Sorghum Improvement and the Genetics of Growth.     Texas A&M University Press. -   Rooney and Aydin, Crop Science, 39; 397-400, 1999. -   Rosyara et al., In: Family-based mapping of FHB resistance QTLs in     hexaploid wheat, Proc. Natl. Fusarium Head Blight Forum, Kansas     City, Mo., 2007. -   Takano et al., Plant Cell, 17(12):3311-3325, 2005. -   Turner et al., Science, 310(5750):1031-1034, 2005. 

1. A method for producing a late flowering or non-flowering hybrid sorghum plant comprising crossing a first early flowering sorghum plant with a second early flowering sorghum plant, wherein each of the first and second early flowering sorghum plants is homozygous recessive for at least one allele contributing to an early flowering phenotype, and wherein the first and second early flowering sorghum plants are not homozygous recessive for the same allele contributing to an early flowering phenotype.
 2. (canceled)
 3. The method of claim 1, wherein crossing comprises crossing a first sorghum plant heterozygous dominant for at least a Ma6 allele with a second sorghum plant homozygous recessive for at least the Ma6 allele.
 4. The method of claim 1, wherein crossing comprises crossing a first sorghum plant homozygous dominant for at least a Ma6 allele with a second sorghum plant homozygous recessive for at least the Ma6 allele.
 5. (canceled)
 6. The method of claim 1, wherein the first or second early flowering sorghum plant is produced by a) mutagenizing a late flowering or non-flowering sorghum plant to produce early flowering progeny comprising an inactive gene in a photoperiod sensing pathway.
 7. The method of claim 6, wherein the gene in a photoperiod sensing pathway is Ma6. 8-9. (canceled)
 10. The method of claim 7, wherein the Ma6 gene comprises a nucleic acid encoding sorghum Prr37. 11-12. (canceled)
 13. The method of claim 1, comprising marker-assisted selection of a progeny plant of the cross comprising at least a first genetic marker genetically linked to a Ma6 allele.
 14. (canceled)
 15. The method of claim 13, wherein the genetic marker genetically linked to the Ma6 allele comprises a nucleic acid encoding a sorghum Prr37 polypeptide.
 16. The method of claim 15, wherein the sorghum Prr37 polypeptide comprises a lysine at position
 166. 17. The method of claim 16, wherein the sorghum Prr37 polypeptide is encoded by a nucleic acid molecule comprising SEQ ID NO:1.
 18. The method of claim 13, wherein the genetic marker is genetically linked to a quantitative trait locus (QTL).
 19. The method claim 18, wherein the QTL is selected from the group consisting of FlrAvgB1, FlrAvgD1, FlrFstG1, F1tQTL-DFG, F1tQTL-DFB, QMa50.txs-A, QMa50.txs-C, QMa50.txs-F1, QMa50.txs-F2, QMa50.txs-H, QMa50.txs-I, QMa1.uga-G, QMa1.uga-D, and QMa5.uga-D.
 20. The method of claim 13, wherein the genetic marker is selected from the group consisting of sequence variants revealed by direct sequence analysis, restriction fragment length polymorphisms (RFLP), isozyme markers, allele specific hybridization (ASH), amplified variable sequences of plant genome, self-sustained sequence replication, simple sequence repeat (SSR) and arbitrary fragment length polymorphisms (AFLP). 21-22. (canceled)
 23. A method of producing an inbred early flowering sorghum plant comprising: a) crossing a late flowering or non-flowering sorghum plant homozygous dominant for Ma5 and Ma6 with an early flowering sorghum plant homozygous recessive for a Ma5 or Ma6 allele; b) inbreeding a F₁ progeny; and c) selecting for an early flowering sorghum F₂ plant homozygous recessive for Ma5 or Ma6 but not homozygous recessive for the same Ma5 or Ma6 allele.
 24. The method of claim 23, wherein the late flowering or non-flowering sorghum plant comprises superior bioenergy properties.
 25. The method of claim 24, wherein the selected early flowering sorghum F₂ plant comprises said superior bioenergy properties.
 26. An early flowering sorghum seed produced by the method of claim
 23. 27. A method of identifying the genotype of a sorghum plant for a Ma5 or Ma6 allele comprising: a) obtaining a sorghum plant; and b) assaying the sorghum plant for a genetic marker genetically linked to the Ma5 or Ma6 allele.
 28. (canceled)
 29. The method of claim 27, wherein the genetic marker genetically linked to an Ma6 allele is a nucleic acid encoding a sorghum Prr37 polypeptide.
 30. The method of claim 29, wherein the sorghum Prr37 polypeptide comprises a lysine at position
 166. 31. The method of claim 30, wherein the sorghum Prr37 polypeptide is encoded by a nucleic acid molecule comprising SEQ ID NO:1. 32-34. (canceled)
 35. The method of claim 3, wherein said Ma6 allele is located on chromosome 6 between coordinates 39379760-42610705. 